JP6103796B2 - コラーゲンゲル収縮促進剤の選択方法 - Google Patents
コラーゲンゲル収縮促進剤の選択方法 Download PDFInfo
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- JP6103796B2 JP6103796B2 JP2010157879A JP2010157879A JP6103796B2 JP 6103796 B2 JP6103796 B2 JP 6103796B2 JP 2010157879 A JP2010157879 A JP 2010157879A JP 2010157879 A JP2010157879 A JP 2010157879A JP 6103796 B2 JP6103796 B2 JP 6103796B2
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Description
(1)メカノセンサーを有する基質に試験物質を添加する工程;
当該メカノセンサーの活性又は発現を測定する工程;及び
当該活性又は発現に基づいて当該試験物質のコラーゲンゲル収縮促進効果を評価する工程、
を含む、コラーゲンゲル収縮促進剤の選択方法。
(2)前記メカノセンサーがp130Casである、(1)に記載の方法。
(3)コラーゲンゲル中の細胞におけるメカノセンサーの活性又は発現を制御する工程を含む、コラーゲンゲル収縮促進方法。
(4)前記メカノセンサーがp130Casである、(3)に記載の方法。
(5)メカノセンサーを有する基質に試験物質を添加する工程;
当該メカノセンサーの活性又は発現を測定する工程;及び、
当該活性又は発現に基づいて当該試験物質の皮膚状態改善効果を評価する工程、
を含む、皮膚状態改善剤の選択方法。
(6)前記皮膚状態が、皮膚の弾性、しわ、たるみ、はりの低下又は創傷である、(5)に記載の方法。
(7)前記メカノセンサーがp130Casである、(5)に記載の方法。
(8)請求項1に記載の方法で選択されたコラーゲンゲル収縮促進剤を含む、メカノセンサー活性制御剤。
(9)前記メカノセンサーがp130Casである、(8)に記載のメカノセンサー活性制御剤。
方法
(1)p130Cas標的RNAi
ヒト真皮線維芽細胞(cell system社)にp130Cas標的siRNAとしてBCAR1-HSS114271、BCAR1-HSS114272、およびBCAR1-HSS114273(全てStealthTM RNAi、Invitrogen社)を製品添付のプロトコールに従い、LIPOFECTAMINETM 2000(Invitrogen社)を用いて導入した(40nM siRNA、2μl LIPOFECTAMINETM 2000)。陰性対象として、同様の手順でStealthTM RNAi Nagetive Control Duplexes(Invitrogen社)を導入した。導入24時間後の細胞を用いてコラーゲンゲルを作製した。
(2)コラーゲンゲル作製
既報(J. Cell Science,102,315 (1992)、J. Invest. Dermatol,93,792 (1989))に準じた方法でコラーゲンゲルを作製した。すなわち、コラーゲンゲルは氷冷下でコラーゲンゲル溶液(新田ゼラチンtypeI-A(3.0mg/ml pH 3))にHEPES(250mM)、DMEM(GIBCO DMEM, low glucose)5倍濃縮溶液、FCS、および精製水を加え、充分に攪拌した。続いてsiRNA導入24時間後の細胞の懸濁液を細胞密度が1.0×105 cells/dishとなるように上記混合溶液に加えた。このとき、コラーゲンは1.5mg/ml、HEPESは25mM、FCSは2.5%となっている。最後にこの線維芽細胞含有コラーゲンゲル溶液をφ3.5cm dishに2mlずつ分注し、37℃で培養することでゲル化させた。
(3)Western blot解析によるゲル包埋細胞中のp130Cas発現解析
(2)の手法によりゲル化させたゲルをdishより剥離し、剥離直後(収縮前)、及び剥離後DMEM(2.5%FCS)中に浮遊させて4日間培養後(収縮4日目)させた後、遠心により細胞が包埋されたゲルを培地から分離した。分離したゲルをRIPA buffer中でガラスホモジナイザーにてすりつぶし、細胞内のタンパク質を抽出した。抽出したタンパク質溶液を90〜100℃で変性させ、SDS−PAGEでサンプルを分離後、Immobilon(PVDF transfer membrane、MILLIPORE)にブロットした。その後、5%スキムミルク/PBS-T(0.1% Tween20/PBS)溶液中でブロッキングした後、一次抗体との反応、およびHRP結合二次抗体との反応を行った。p130Cas(Total)の検出にはp130Cas抗体(BD Transduction Labs社)を一次抗体として用いた。結果を図1Aに示す。
(4)コラーゲンゲル収縮の評価
(2)の手法によりゲル化させたゲルをdishより剥離し、DMEM(2.5%FCS)中に浮遊させて培養し、経時的に撮影したゲルの写真を画像解析し、直径を測定した。ゲルの収縮は剥離前の直径に対する減少率で評価した。DMEM培養コラーゲンゲルの写真を図1Bに、ゲル減少率の測定結果を図1Cに示す。
p130Cas標的RNAiにより、ゲル収縮評価期間中(4日間)メカノセンサーp130Casの発現は阻害されており(図1A)、またコラーゲンゲル収縮が抑制された(図1B、C)。よって、メカノセンサーが真皮線維芽細胞のコラーゲンゲル収縮活性に関与していることが示され、メカノセンサーの発現又は活性を制御することによりコラーゲンゲル収縮を促進し、また皮膚状態を改善することができることが示唆された。
(1)試験物質
試験物質として、ミシマサイコ(Bupleurum falcatum L.)、およびオランダガラシ(Nasturtium officinale)の水抽出物を用いた。
ミシマサイコ(Bupleurum falcatum L.)の根(新和物産)40gに、400mLの水を加え、70℃で5時間抽出後、ろ過し、ろ液を濃縮して、ミシマサイコ水抽出物11.8gを得た。
オランダガラシ(Nasturtium officinale)(ベトナム産)100gに、1000mlの水を加え、70℃で5時間抽出後、ろ過し、ろ液を濃縮して、オランダガラシ抽出物23.11gを得た。
なお、ミシマサイコエキス及びオランダガラシエキスは、それぞれ特開2001-39850号公報及び特開2006-316050号公報に開示されるように、ハリ、タルミ改善剤として知られている。
フィブロネクチンでコーティングしたシリコンエラストマー製のStretch chamber(ストレックス社)にヒト真皮線維芽細胞を2.2×104cells/cm2で播種し、翌日、終濃度が0.001(w/v)%(乾燥固形換算重量%)となるように上記で調製した各植物エキスあるいはコントロールとして等量の10%エタノールを加えたDMEM(FCS不含)培地と交換し、さらに3日間培養を行った。
細胞伸展装置STREX(ストレックス社)に正常ヒト表皮角化細胞(NHEK)を播種したチャンバーをセットし、伸展率20%で伸展刺激を負荷した。2分間負荷した後、p130Casのリン酸化(活性化)状態を保持しながら細胞からタンパク質を抽出するため、phosphatase inhibitorを含む抽出bufferであるPhosphoSafe Extraction Reagent(Novagen社)を200μlずつチャンバーに加え、室温で5分間インキュベーションした。
抽出したタンパク質溶液を90〜100℃で変性させ、SDS-PAGEでサンプルを分離後、Immobilon(PVDF transfer membrane、MILLIPORE)にブロットした。その後、5%スキムミルク/PBS-T(0.1% Tween20/PBS)溶液中でブロッキングした後、一次抗体との反応、およびHRP結合二次抗体との反応を行った。リン酸化p130Casの検出にはPhospho-p130Cas (Tyr165) Antibody(Cell Signaling社)を一次抗体として用いた。各群から検出されたバンドの強度を、Control(エタノール添加群)の非伸展刺激サンプル(Non treated)での強度を1.00とした相対値(arbitrary units)として算出し、各群でのp130Casのリン酸化(活性化)レベルを評価した。
結果を図2に示す。ゲルの伸展刺激に応じてリン酸化p130Cas(p-p130Cas)の量が増加しており、伸展刺激によりp130Casが活性化されたことが示された。伸展刺激に加えてミシマサイコエキス、オランダガラシエキスを添加した場合、p-p130Casはさらに増加したことから、活性化がより増強されたことが分かった。すなわち、ミシマサイコエキス、オランダガラシエキスの水抽出物は、いずれもメカノセンサーp130Casの活性を増強させた。
ヒト皮膚真皮線維芽細胞(cell system社)を用いた。コラーゲンゲルは氷冷下でコラーゲンゲル溶液(新田ゼラチンtypeI-A(3.0mg/ml pH=3))にHEPES(250mM)、DMEM
(GIBCO DMEM, low glucose)5倍濃縮溶液、FCS、および精製水を加え、充分に攪拌した後、終濃度が0.001(w/v)%(乾燥固形換算重量%)となるようにミシマサイコエキス、あるいはコントロールとして等量の10%エタノールを加えた。続いてヒト線維芽細胞の懸濁液を細胞密度が2.5×104cells/wellとなるように上記混合溶液に加えた。終濃度で、コラーゲンは1.5mg/ml、HEPESは25mM、FCSは2.5%となっている。最後にこの線維芽細胞含有コラーゲンゲル溶液を48穴プレートに600μl/wellずつ分注し、37℃で培養することでゲル化させた。ゲル化後、プレートよりゲルを剥離し、DMEM(2.5%FCS)中に浮遊させて培養し、2日後、ゲルの直径および重量を測定した。エキスのゲル収縮促進効果は、各エキスを添加したゲルの直径および重量についてコントロールを100とした場合の相対値で評価した。
Claims (3)
- p130Casを有する基質に試験物質を添加する工程;
p130Casタンパク質の活性、又はp130Casタンパク質、その遺伝子若しくはmRNAの発現を測定する工程;及び
当該活性又は発現に基づいて当該試験物質のコラーゲンゲル収縮促進効果を評価する工程、
を含む、コラーゲンゲル収縮促進剤の選択方法。 - p130Casを有する基質に試験物質を添加する工程;
p130Casタンパク質の活性、又はp130Casタンパク質、その遺伝子若しくはmRNAの発現を測定する工程;及び、
当該活性又は発現に基づいて当該試験物質の皮膚状態改善効果を評価する工程、
を含む、皮膚状態改善剤の選択方法。 - 前記皮膚状態が、皮膚の弾性、しわ、たるみ、はりの低下又は創傷である、請求項2に記載の方法。
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